Publications by authors named "Prasada K Shama"

2 Publications

  • Page 1 of 1

γ-radiation induces cellular sensitivity and aberrant methylation in human tumor cell lines.

Int J Radiat Biol 2011 Nov 7;87(11):1086-96. Epub 2011 Sep 7.

Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Karnataka, India.

Purpose: Ionizing radiation induces cellular damage through both direct and indirect mechanisms, which may include effects from epigenetic changes. The purpose of this study was to determine the effect of ionizing radiation on DNA methylation patterns that may be associated with altered gene expression.

Materials And Methods: Sixteen human tumor cell lines originating from various cancers were initially tested for radiation sensitivity by irradiating them with γ-radiation in vitro and subsequently, radiation sensitive and resistant cell lines were treated with different doses of a demethylating agent, 5-Aza-2'-Deoxycytidine (5-aza-dC) and a chromatin modifier, Trichostatin-A (TSA). Survival of these cell lines was measured using 3-(4, 5-Dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium (MTT) and clonogenic assays. The effect of radiation on global DNA methylation was measured using reverse phase high performance liquid chromatography (RP-HPLC). The transcription response of methylated gene promoters, from cyclin-dependent kinase inhibitor 2A (p16(INK4a)) and ataxia telangiectasia mutated (ATM) genes, to radiation was measured using a luciferase reporter assay.

Results: γ-radiation resistant (SiHa and MDAMB453) and sensitive (SaOS2 and WM115) tumor cell lines were examined for the relationship between radiation sensitivity and DNA methylation. Treatment of cells with 5-aza-dC and TSA prior to irradiation enhanced DNA strand breaks, G2/M phase arrest, apoptosis and cell death. Exposure to γ-radiation led to global demethylation in a time-dependent manner in tumor cells in relation to resistance and sensitivity to radiation with concomitant activation of p16(INK4a) and ATM gene promoters.

Conclusion: These results provide important information on alterations in DNA methylation as one of the determinants of radiation effects, which may be associated with altered gene expression. Our results may help in delineating the mechanisms of radiation resistance in tumor cells, which can influence diagnosis, prognosis and eventually therapy for human cancers.
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http://dx.doi.org/10.3109/09553002.2011.605417DOI Listing
November 2011

A novel human CRYGD mutation in a juvenile autosomal dominant cataract.

Mol Vis 2010 May 22;16:887-96. Epub 2010 May 22.

Department of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal, Karnataka state, India.

Purpose: Identification of causal mutation in the crystallin, connexin, and paired box gene 6 (PAX6) genes associated with childhood cataract in patients from India.

Methods: In this study, forty eight members from seventeen families and 148 sporadic cases of childhood cataract were evaluated. Clinical and ophthalmologic examinations were performed on available affected and unaffected family members. Samples of genomic DNA were PCR amplified to screen for mutations in the candidate genes viz., alpha-A crystallin (CRYAA), beta- B2 crystallin (CRYBB2), gamma-A crystallin (CRYGA), gamma-B crystallin (CRYGB), gamma-C crystallin (CRYGC), gamma-D crystallin (CRYGD), gap junction alpha-3 (GJA3), gap junction alpha-8 (GJA8), and PAX6 based on polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) analysis. Samples showing any band mobility shift were subjected to bidirectional sequencing to confirm the variation. Co-segregation of the observed change with the disease phenotype was further tested by restriction fragment length polymorphism (RFLP) for the appropriate restriction site.

Results: DNA sequencing analysis of CRYAA, CRYBB2, CRYGA-D, GJA3, GJA8, and PAX6 of the affected members of a family (C-35) showed a novel heterozygous missense mutation C>A at position 229 in CRYGD in three affected members of family C-35 with anterior polar coronary cataract. This variation C229A substitution created a novel restriction site for AluI and resulted in a substitution of highly conserved arginine at position 77 by serine (R77S). AluI restriction site analysis confirmed the transversion mutation. Analysis of the available unaffected members of the family (C-35) and 100 unrelated control subjects (200 chromosomes) of the same ethnic background did not show R77S variation. Data generated using ProtScale and PyMOL programs revealed that the mutation altered the stability and solvent-accessibility of the CRYGD protein.

Conclusions: We describe here a family having anterior polar coronary cataract that co-segregates with the novel allele R77S of CRYGD in all the affected members. The same was found to be absent in the ethnically matched controls (n=100) studied. Interestingly the residue Arg has been frequently implicated in four missense (R15C, R15S, R37S, and R59H) and in one truncation mutation (R140X) of CRYGD. In two of the reported mutations Arg residues have been replaced with Serine. This finding further expands the mutation spectrum of CRYGD in association with childhood cataract and demonstrates a possible mechanism of cataractogenesis. Screening of other familial (n=48) and sporadic (n=148) cases of childhood cataract, did not reveal any previously reported or novel mutation in the candidate genes screened.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875257PMC
May 2010